SEPARATION OF DNA FRAGMENTS BY CAPILLARY ELECTROPHORESIS USING REPLACEABLE LINEAR POLYACRYLAMIDE MATRICES

The use of low percent (1.5-6%T) replaceable linear polyacrylamide (LPA) network matrices for rapid separation of double-stranded DNA fragments was explored. Separations of fragments ranging from 20 to 23 000 base pairs were readily achieved. Typically, 4 . 10(6) theoretical plates/m were obtained in less than 30 min. Short separation times under 2 min were also possible, using the DNA intercalating dye, ethidium bromide, along with high electric fields. The high resolving power of linear polyacrylamide was demonstrated in the separation of two fragments which differ by a single base pair (123/124 base pairs) using 6%T LPA and ethidium bromide intercalation. This LPA composition allowed for the possible single base-pair resolution of dsDNA fragments up to 300 base pairs in length. Several concentrations of the linear polyacrylamide for different-ranges of fragment lengths have been employed. In addition, replaceable LPA offers the advantage of a fresh separation matrix for each run, thus overcoming column stability problems and minimizing needs for sample cleanup. Electroosmotic flow was substantially reduced using stable capillary coatings, which were required for obtaining high efficiencies and good reproducibility.